666-15 is a selective CREB inhibitor with an IC50 of 81 nM. 666-15 can effectively inhibit the growth of breast cancer.

CREB:81 nM (in HEK 293T cells)

METHODS: 666-15 was used to treat OA-like chondrocytes at a concentration of 62.5 to 500nM, and the viability of 666-15 on OA-like aged guinea pig chondrocytes was studied.
RESULTS p-CREB1 protein levels were significantly reduced and cell viability of aged guinea pig chondrocytes was significantly increased. [1]
METHODS: To investigate the biological effects of CREB inhibition, SySa cells were incubated with increasing concentrations (0.13-5 μM) of 666-15 and subjected to MTT assay.
RESULTS 666-15 significantly inhibited SySa cell viability in a dose-dependent manner (IC50: 0.36-1.72 μM). [2]

METHODS: Mice were randomly assigned to different groups, including normal group, sham operation group, ACLT+solvent control group, and two different doses of 666-15 treatment groups (ACLT+666-15 low-dose group, 5 mg/kg; ACLT+666-15 high-dose group, 10 mg/kg, intraperitoneal injection, once a week for six weeks).
RESULTS 666-15 treatment can alleviate joint degeneration, reduce the levels of cartilage degradation markers such as CTX-II, reduce synovial inflammation, and has no effect on the body weight of mice, indicating its safety. [1]

HEK 293T cells in a 10 cm plate were transfected with pCRE-RLuc (6 μg) with Lipofectamine2000 following the manufacturer's instructions. Three hours after transfection, the cells were collected and replated into 96-well plates at ～10?000 cells/well. The cells were allowed to attach to the bottom of the plates overnight. The cells were then treated with different concentrations of different compounds for 30 min when forskolin (10 μM) was added to each well. The cells were incubated for further 5 h before cell lysis using 1× 30 μL Renilla luciferase lysis buffer. An amount of 5 μL of the lysate was combined with 30 μL of benzyl-coelenterazine solution in PBS (pH 7.4, 10 μg/mL). The protein concentration in each well was determined. The Renilla luciferase activity was normalized to protein content in each well and expressed as relative luciferase unit/μg protein (RLU/μg protein). The IC50 was derived from nonlinear regression analysis of the RLU/μg protein–concentration curve [1].

Each 6- to 8-week old BALB/c nude mouse was inoculated subcutaneously at the right flank with MDA-MB-468 cells (5 × 10^6) in 0.1 mL of HBSS with Matrigel (1:1) for tumor development. When the tumor volume reached approximately 100 mm3, the mice were randomized to be treated with either vehicle or 666-15 at 10 mg/kg. 3i was dissolved in 1% N-methylpyrrolidone (NMP), 5% Tween-80 in H2O. The dosing solution was prepared weekly. The mice were treated once a day for 5 consecutive days a week, and the treatment lasted for 5 weeks. During the treatment, the tumor size and body weight were measured 2–3 times a week. The tumors were measured in two dimensions using a digital caliper, and the volume was expressed in mm3 using the formula V = 0.5ab2, where a and b represent the long and short diameters of the tumor, respectively. The tumor volume was normalized to the initial tumor volume at the time of the first treatment [1].